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High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.